RESEARCH OF THE INFLUENCE OF AIR TRANSPORTATION ON BLOOD SAMPLE QUALITY

In air freight industry, blood samples are classified as time and temperature sensitive biologically produced pharmaceuticals. To determine the level of influence that the handling processes and air transportation have on blood sample quality, a research has been conducted through transportation of whole blood samples on two European scheduled routes. Two shipping models were defined: the standard one without defined transportation temperature regime and the controlled one, where transportation is conducted under appropriate temperature regime. The blood samples were packed and transported respecting all relevant national and international regulations. The analysis was conducted and the results compared to control sample kept in the laboratory. Significant changes were identified on all components analyzed after crosschecking with the control sample.


INTRODUCTION
The Act on Blood and Blood Components [1] (Croatian Parliament, 30 June 2006, Official Gazette No. 79/06) Chapter VII Export and import of blood and blood samples, in Articles 24 and 25 has issued a prohibition of exporting and importing blood and blood components into and out of the Republic of Croatia.At the same time, both Articles stipulate also special circumstances in which the respective Ministry can exceptionally give clearance for the export, i.e. import of blood and blood components.Unlike blood components, blood samples are relatively often subject of transport, mainly air transport, in order to implement testing or research.All samples have to be non-reactive to the markers of transfusion-transmitted infection diseases.For the needs of the respective research there has been a request to obtain clearance to use blood samples of non-related volunteer blood donors who have been informed about the research and have voluntarily given their written consent to participate in the research.The request for implementation of the research was submitted to the Ethical Committee of the Croatian Institute of Transfusion Medicine (Hrvatski zavod za transfuzijsku medicinu, HZTM).By issuing a positive opinion of the Committee the legal assumptions were met to start the research project.
According to a defined protocol the blood samples were extracted from the pre-donation bags with a documented prior consent of the donor.Apart from the mentioned Act, the research procedure recognised also all the other valid acts and regulations related to the distribution of blood and blood components in international transport [2], [3].The distribution channel which includes also the transportation has been regulated by the national and international documents on good manufacturing, storage and distribution practice [4], [5], [6].The conditions of air transport handling have been defined by the documents of the Interna-tional Air Traffic Associations (IATA) and the International Civil Aviation Association (ICAO).
In compliance with the Act on Blood and Blood Components, Regulations on the Blood Component Traceability System and Monitoring of Serious Adverse Events and Serious Adverse Reactions (NN 63/07), Regulations on Quality Assurance of Blood and Blood Components in Medical Institutions (NN 80/07), Regulations on Special Technical Requirements for Blood and Blood Components (NN 80/07) and finally the quality assurance procedures, after identification, all the donors were tested to determine the level of haemoglobin, they underwent a medical check and had an interview with the authorized physician who was in charge of examining the donors.The physician informed them about the planned research and asked them to sign the form of consent.The person who performs venepunction used the standard procedure following venipuncture to fill the pre-donation bags.
The blood from the pre-donation bag served to isolate the sample for the regulated test and to isolate the first amount of blood which may have contained bacteria contamination from the skin surface at the point of puncture.All the procedures from the entry of the donor, check and venipuncture were done in compliance with the stipulated work instructions that are part of the quality system documentation of HZTM.The samples for immune-haematological test and test for markers of transfusion-transmitted infection diseases were sent to the laboratories where they were subjected to the tests subscribed according to the professional rules and stipulated work instructions.All samples for this research were sent to the Department of Quality Assurance and Control and were tested for the desired haematological parameters in accordance with the respective work instructions.

DEFINITION OF PARTICIPANTS, OBJECTIVES, AND DYNAMICS OF THE RESEARCH PROJECT
During observation of the technological process of handling biological samples in air transport, which means also the realisation of the transport task, it is necessary to distinguish the subjects i.e. participants within the very process, to define their role and to determine the responsibility areas as well as the transition points [7].The responsibility of each participant has been defined in the documents that regulate the relations regarding the transport of perishable and temperature sensitive shipments [8] (IATA, Perishable Cargo Regulations, 8 th Edition), as well as in the documents that regulate the transport of dangerous goods [9] (IATA, Dangerous Goods Regulations, 50 th Edition) since biological samples, according to their character may be subject of the latter regulation.In the segment of responsibility, the technological process of transporting biological samples has been passing through four different areas of stakeholder's responsibility in interdependence and interaction.Thus, in the realization of the project, in this concrete case, there are the shipper, the logistic operator, the carrier and the consignee.Starting from the fact that the basic objective was the intention of proving the significant impact of air transport environment on the quality of blood samples, the following project subobjectives were defined: -Realization of the legal obligations as preconditions to start a research project.-Extraction of blood samples at HZTM. -Haematological analysis of extracted samples at HZTM. -Realization of the export transportation task.
-Realization of the import transportation task.
-Haematological analysis of the extracted samples at HZTM. -Systematization and publishing of the collected results.
The conditions under which a blood sample remains in its original quality are strictly defined.A control sample is kept under controlled conditions at +4°C during storage at HZTM.The temperature regime required for transportation of blood samples is +2°C to +8°C defined as cold.This temperature range is considered as controlled transportation conditions.In order to subject the samples to realistic handling and transportation conditions, a part of the samples prepared is transported in standard transportation conditions referred to as uncontrolled conditions.
The project was realized according to the pre-defined protocol through several transport tasks.The number of deliveries in the project was determined by the conclusions deduced from each previous one.The tasks of each single stakeholder in the realization process have been precisely determined by activities in the given time and at the given locality.A detailed presentation of the realization was framed in the form of Standard Operating Procedure 1 .The agreement among stakeholders within the defined technological process regarding technology of the transport task realisation, time and location of performance as well as points of responsibility transition represents the precondition for successful realization.The clarity of the defined objective, sub-objectives, tasks and procedures planned in every segment of the project realization were intended to achieve high level of success in realization.
For the data on the blood sample quality collected during the transportation procedure to be referencecomparable, the realization was carried out according to the model presented in Diagram 1.This diagram shows the process of sending the blood samples to two different destinations in two different regimes.The criteria for carrier selection are a total of 1,052 blood sample shipments transported from the Zagreb Airport during the year 2007 [10].The routes and destinations were selected according to the same criteria thus applying regular handling and transportation flows applicable at the Zagreb airport.
The first model is denoted as Model A and represents the realization of the transportation task using a scheduled flight from Zagreb via Vienna to Brussels and back, whereas in the second Model (Model B) London was planned as the international destination.

REALIZATION OF TRANSPORT TASK AND COLLECTED DATA PROCESSING
The data collected from the realization were systematised in two groups.The first group of data comprised observations and measurements recorded during the realization of the technological process of transport, and the second one included observations regarding the phenomena related to the changes in the structure and quality of blood sample as subject of transport.The realization was carried out according to a pre-defined and from all the stakeholders accepted  SOP, with a more concrete presentation of the realization of one of the models (Table 1).
The second group of observations referred to the phenomena related to the change in the structure and quality of blood sample as the subject of transport.Vein bloods of donors were extracted from a test tube with K2EDTA 5 (5.4 mg) a' 3 mL.Five identical samples each were taken from 37 donors.The samples were distributed in three groups.One remained at HZTM (controlled conditions in a refrigerator), the second was packaged for transport via Vienna to Brussels, and the third via Vienna to London in compliance with the pre-defined algorithm.Among samples that were grouped as reference group that remained at HZTM the following parameters were measured immediately upon drawing the blood: number of leucocytes, number of leucocytes of neutrophyl line, number of erythrocytes, haemoglobin amount, value of hematocrit, mean volume of erythrocytes, mean content of haemoglobin in erythrocytes, mean concentration of haemoglobin in erythrocytes, number of thrombocytes, mean volume of thrombocytes.With all the performed tests the lots of microcuvettes and control bloods used in the measurements were recorded.The abovementioned parameters and the haemolysis of erythrocytes have been measured in groups that after the transport were returned to HZTM, which was done immediately upon return.Then the measurement of the control group that was stored in controlled conditions in the HZTM refrigerator was repeated.
An example of the presentation of measurements and processing of the obtained results for one of the groups (controlled transport conditions to London) and the results of haemolysis are presented in Table 3.The presented values of hematocrits in the percentage and in total amount of haemoglobin were measured just after drawing blood.The following column shows the values of haemoglobin measured in supernatant after the blood samples have been returned from London.and finally the calculation of the amount of haemolysis expressed in percentage has been presented.At the bottom of the table the lots of microcuvettes and control bloods used in the measurements are presented.
The obtained results were processed statistically, according to groups and compared.For each group of samples the mean value and standard deviation have been calculated for each required parameter.A calculation of t-test has also been performed showing whether there is statistical significance in the differ-ence of the obtained results.The statistically significant difference is considered to be if the result of t-test is < 0.05.
The presented results show that there was a statistically significant difference in the comparison of results obtained from blood which were subjected to the process of transportation, either in controlled or in non-controlled conditions, and those stored at HZTM with the results obtained from the blood samples measured immediately upon drawing blood.
Table 4 shows the results of the statistical processing of the data obtained by measuring the amount of erythrocytes haemolysis.
Comparing the results of the transported blood with the one stored at HZTM, there was also statistically significant difference found in all the determined parameters.There was also statistically significant difference noticed in the results among samples subjected to transport in controlled and non-controlled conditions on the flights to Brussels and London.

RECORDINGS OF MEASUREMENT INSTRUMENTS DURING TRANSPORT TASK REALIZATION
During the realization of the transport task in order to determine the temperature profile of realization [11], [12], the temperature measurement instruments have been used 23 .Temperature monitoring has been done in such a way that for every packaging of blood samples two instruments each have been used; one being located on the packaging itself on the outside, and the other one inside the packaging (Figure 2).
External measuring instrument was installed on top of the box in order to measure the temperature that develops during sample handling.The latter can be understood as reference in relation to the measured values of ambient temperature for the time when during the processing the shipment was exposed to ambient atmosphere influences.The interior measurement instrument was installed after having set a layer of paper tissues around the primary packaging of the sample.In this way the instrument set within the packaging was not in direct contact with the samples, but the temperature recordings were showing the temperature profile within the packaging.The paper tissues were used as an absorbing and cushioning material within the package in compliance with the provisions   ).Temperature recordings of measurement instruments during transport were considered at 11 reference points.These points in the process of transportation may be considered as significant since they are characterized by a high level of interaction of all the included process stakeholders, emphasised significance of responsibility transfer from one stakeholder to another, and the specific environment in which the transport object is found.Table 5 shows the defined steps as control points in monitoring the recordings of measurement instruments.
Based on the temperature recordings through the mentioned steps, the temperature profile for each transport model was formed.In order to form the temperature profile for the entire year, the recommendation is to make a summer and a winter temperature profile 25 .The concrete case refers to the realisation of the project during the month of March which makes it possible to deduce a conclusion acceptable for the winter profile.Graph 1 shows the temperature recording during the realisation of the transport task from model A. The temperature requirement for both models in controlled conditions is the transport at a temperature regime ranging from +2°C to +8°C (denoted by a green line in Graphs 2 and 4), whereas the noncontrolled conditions mean warehousing in standard ambient conditions.

CONCLUSION
By analysing the samples before and after the transport in air freight and by comparing them with the control sample stored in HZTM at controlled tempera-ture conditions at +4°C, considerable changes were noticed on all observed components.The changes presented have been brought directly into context with the conditions to which the samples were subjected during handling and transportation.While observing the efficiency of the packaging designed for this kind of commodity it could be concluded that the application of the cooling elements within the same type of packaging is more efficient in achieving stable temperature regime.At the same time, it could be concluded that the absence of preconditioning of the commodity and packaging could result in the failure to achieve required temperature conditions or +2°C to +8°C like in the concrete case.Figure 3 shows the existence of a temperature extreme with the peak at +6.5°C.This is an interesting example because it has been recorded within the package which was not equipped with the cooling elements.The reasons for this phenomenon could be found in the fact that the loading position in an aircraft might have an impact on the temperature profile during transportation (loading on positions close to the compartment door could result in lower temperatures).The handling process duration or exposures to environmental conditions as well as the lack of appropriate storage infrastructure might also influence the stability of the temperature regime.Following the gained results after sample analysis it can be concluded that air transportation will have a considerable influence on the blood sample quality.The repetition of the research is thus recommended during summer months when higher atmospheric temperatures are expected having a greater impact on temperatures recorded during the experiment.It could be assumed that higher exposure temperatures might result in more considerable impact on blood sample quality.

Temp. 1 -Figure 3 -Figure 4 -Figure 5 -
Figure 3 -Temperature profile of transport model A with warehousing in standard warehousing conditions

Figure 6 -
Figure 6 -Temperature profile of transport model B with warehousing in controlled warehousing conditions at +2°C to +8°C

Table 1 -
Module A, realization of the transport task in the project Majić, I. Jukić, T. Vuk, S. Pavlin: Research of the Influenceof Air Transportation on Blood Sample Quality nd Transport is performed on Zagreb -Vienna --London route Figure 1 -Transport task realization model Promet -Traffic&Transportation, Vol. 23, 2011, No. 6, 431-442

Table 2 -
Example of presentation of measurement results and processing of one of the analysis parameters (number of erythrocytes)

Table 3 -
Values of hematocrits, total amount of haemoglobin, amount of haemoglobin in supernatant, and the results of haemolysis

Table 4 -
Results of statistical processing of the haemolysis amountHatched area designates the place of putting paper tissues used as material for asorbing and prevention of shifting of samples within the exterior packaging.

Table 5 -
Reference steps in technological process of project realization with recorded temperature